The relationship between exhaled carbon monoxide and human neutrophil function in the Japanese general population

We have evaluated the relationship between exhaled carbon monoxide (CO) level and neutrophil‐related functions such as reactive oxygen species (ROS) production capability, phagocytic activity and serum opsonic activity in the general population. Serum opsonic activity was determined by measuring the effects of serum on neutrophil ROS production capability using lucigenin‐ and luminol‐dependent chemiluminescence (LgCL, LmCL). LgCL is associated with the detection of O₂^?, whereas LmCL mainly detects H₂O₂ and HOCl, which are higher reactive oxygen radicals. In females, exhaled CO level was found to have positive associations with ROS production capability and LgCL. However, the opposite tendency was seen between exhaled CO level and LmCL in both genders. This result suggests that neutrophil ROS production in females may have contributed to oxidative stress, which led to the increases in intrinsic CO and exhaled CO consequently. Such changes then may have inhibited the process of changing reactive oxygen radicals into higher oxidizing potential levels. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of summer training camp on serum opsonic activity and plasma cytokine levels in female long‐distance runners

In a summer training camp, strenuous physical exercises are carried out to improve endurance ability. The effects of the training stress on the immune system have been studied extensively. However, less attention has been paid to non‐specific immunological changes. Serum opsonic activity (SOA) is a more direct and suitable indicator of non‐specific humoral immunity. In this study, we used the luminal‐dependent chemiluminescence (LmCL) to measure reactive oxygen species generation from pooled human neutrophils as an indicator of SOA. We also measured plasma myeloperoxidase (MPO) and plasma cytokine levels. Twenty‐two female college runners took part in this study after giving their written informed consent. There was no significant difference in the physical characteristics and serum enzyme levels. However, significant differences were observed in the changes of blood property and plasma cytokine levels. According to MPO levels, neutrophils in vivo may be deactivated during the camp. Positive correlations between fluctuations of pro‐ and anti‐inflammatory cytokine levels were observed in this study. According to the results of peak time and peak height of LmCL, SOA seems to increase at first qualitatively and then quantitatively during the summer camp. The increased SOA level may compensate for the decrease in neutrophil activity. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of a 21 day metabolic study on serum opsonic activity in female college students, assessed by a chemiluminescence technique.

A metabolic study to evaluate nutritional balance, in which subjects have to eat all the foods supplied and maintain a set schedule, is thought to be stressful for the subjects. The effects of stress on the immune system have been studied extensively. However, there are no studies of non-specific immunological changes during a metabolic study. Serum opsonic activity (SOA) is a direct and suitable indicator of non-specific humoral immunity. In this study, we used luminol-dependent chemiluminescence (LmCL) to measure reactive oxygen species (ROS) generation from pooled human neutrophils as an indicator of SOA. We also measured serum immunoglobulin levels and plasma myeloperoxidase (MPO) activity. Eleven female college students took part in this 21 day metabolic study after giving their written informed consent. The results obtained suggest that the metabolic study has almost no effect on immunoglobulin levels. According to MPO levels, neutrophils in vivo may be deactivated to some minor extent. In contrast to these results, peak time (PT) and peak height (PH) of LmCL were changed significantly during the metabolic study. In conclusion, SOA increased during the 21 day metabolic study. There was no significant correlation between SOA and serum immunoglobulin levels on any of the study days.     

Production of chemokines and reactive oxygen species by human neutrophils stimulated by Helicobacter pylori

Background. Bacteria have different characteristics in stimulation of human neutrophils to produce reactive oxygen species (ROS) and chemokines. This study examined the ability of Helicobacter pylori to induce production of ROS and chemokines by human neutrophils. Methods. H. pylori strains (1.5 × 10⁸ CFU/ml) were cocultured with 5 × 10⁴ neutrophils isolated from healthy subjects. Samples were incubated with human serum with or without IgG antibodies to H. pylori. ROS production was measured using luminol‐dependent chemiluminescence (LmCL), and the concentrations of chemokines (IL‐8, RANTES, MIP‐1α and MCP‐1) were measured by ELISA. Results. The mean of the highest LmCL (peak height; PH) value stimulated by H. pylori was 3318 in the absence of serum. PH increased to 4687 when incubated with anti‐H. pylori antibody‐positive sera (p < .001) but antibody‐negative sera did not affect LmCL response. The mean final concentration of IL‐8 produced in the absence of serum was 142.6 pg/ml. Increased IL‐8 production was seen by addition of antibody positive serum (p < .01). IL‐8 production was not significantly correlated with production of ROS. On the other hand, H. pylori stimulation did not induce neutrophil production of RANTES, MIP‐1α or MCP‐1. Conclusions. H. pylori was capable of inducing IL‐8 production by human neutrophils, but not C‐C chemokines. Production of C‐X‐C dominant chemokine by neutrophils is consistent with the pathological characteristics of H. pylori‐induced gastritis, where persistent neutrophil infiltration is present.     

Effects of 6 months of endurance training on neutrophil functions to produce reactive oxygen species and mental states in male long‐distance runners

To clarify changes of neutrophil functions, mental conditions and relationships among them, 19 male elite long‐distance runners participated in this study for 6 months. Examinations, with informed consent, were carried out once a month. According to the results of physical characteristics, it was thought that training intensity was reduced after the main race, Hakone‐Ekiden. Neutrophil functions were estimated by indices of reactive oxygen species production, determined by luminol‐ and lucigenin‐dependent chemiluminescence (LmCL and LgCL, respectively) and cytochrome c reduction methods. The peak times (PT) in LmCL and LgCL (LgPT) were most prolonged in January and December, respectively. The peak heights (PH) in LmCL (LmPH) were enhanced in February. Decreased levels of negative categories in the profile of mood state (POMS) questionnaire and the total mood state (TMS) of POMS were observed in February without significance. Correlation analysis using measured values revealed significant negative correlation between LmPH and negative categories in POMS; however, these correlations were possibly a mere appearance, caused by personal differences. After eliminating personal differences, LgPT correlated positively to depression (p < 0.05), anger (p < 0.05), fatigue (p < 0.01) and TMS (p < 0.05). These results suggest that the mean time from the recognition of foreign matter to the maximum production of superoxide from neutrophils is prolonged in the mentally suppressed conditions found under continuous physical training. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of glutamine supplementation on neutrophil function in male judoists

Glutamine is an important amino acid for immune function. Though high intensity and prolonged exercise decreases plasma glutamine concentration and causes immune suppression, the relationship between neutrophil functions and glutamine has not yet been found. The purpose of this study was to investigate the impacts of glutamine supplementation on neutrophil function. Twenty‐six male university judoists were recruited. Subjects were classified into glutamine and control groups. The glutamine group ingested 3000 mg of glutamine per day and the control group ingested placebo for 2 weeks. Examinations were performed at the start of preunified loading exercise (pre‐ULE), then 1 and 2 weeks after ULE (post‐ULE). Reactive oxygen species (ROS) production, phagocytic activity, serum opsonic activity and serum myogenic enzymes were measured. Differences between the levels obtained in pre‐ULE and post‐ULE for the two groups were compared. In the glutamine group, ROS production activity increased 1 week after ULE, whereas it was not observed in the control group (P < 0.001). Though myogenic enzymes increased significantly after ULE (P < 0.001), the glutamine group remained unchanged by supplementation during ULE. Glutamine supplementation has prevented excessive muscle damage and suppression of neutrophil function, especially in ROS production activity, even during an intensive training period. Copyright © 2013 John Wiley & Sons, Ltd.     

Serum requirements necessary for the opsonophagocytosis ofescherichia coli O73:K92:H1

TheEscherichia coli O73:K92:H1 serotype, which possesses a capsular antigen immunochemically similar to the capsule of the group C meningococcus, is demonstrated in this study to be resistant to phagocytosis by normal human PMNs and serum and to be dependent upon immune antibody and presumably the classical complement pathway for opsonization. Using both a luminol-dependent chemiluminescence assay and an in vitro bactericidal system, we examined, in both the absence and presence of complement, the opsonic activity of IgM ang IgG antisera. Of the various antisera tested, only those sera cross-reactive with the K92 capsular antigen were found to be opsonic both in vivo and in vitro, while somatic O or lipid A antisera demonstrated no activity. In in vitro studies with capsular IgM and IgG antisera, only IgG demonstrated opsonic activity without complement, whereas IgM required complement for opsonization of O73:K92:H1. These data demonstrate that antisera directed toward capsular antigens are opsonic for this phagocytosis-resistantE. coli, and that complement is a necessity for opsonization in the absence of sufficient capsular IgG antibodies.     

Mechanisms of the mediated neutrophil reactivity to Escherichia coli peptidoglycan

The opsonic properties of normal serum with respect to E. coli peptidoglycan was studied under the actual conditions of the oxygen-dependent metabolism of neutrophils. In the course of the differentiated study of the influence of antibodies, the classical and the alternative cascades of complement the serum was heated, treated with ethylenediaminetetraacetate and ethylene glycol tetraacetate, exhausted in the cold. In serial experiments the opsonic activity of purified fibronectin was studied. The indirect reactions were shown to be the leading mechanisms of the neutrophil-stimulated activity of E. coli peptidoglycan. IgG was found to be in the center of the opsonic cooperation and thus to determine the quantitative manifestation of the total phenomenon. Complement proved to be of lesser importance; depending on the conditions of the experiment, the activation of complement occurred by the alternative way (after the removal of antibodies) or the classical way (whole serum). The actual contribution of IgG-independent and complement-independent opsonins was insignificant. Fibronectin in physiological concentrations showed no opsonic activity.     

Serum opsonic deficiency produced by Streptococcus pneumoniae and by capsular polysaccharide antigens.

The opsonic requirements for phagocytosis of S. pneumoniae types 6, 7, 18, and 23 were determined in normal and C2 deficient serum, and in normal serum chelated with magnesium ethyleneglycoltetraacetic acid. All four strains were effectively opsonized via the alternative complement pathway, a finding suggesting that the capsular polysaccharides of these strains activated complement via the alternative pathway. Since bacteremic pneumococcal disease is often associated with circulating capsular polysaccharide, it was considered that this cellular component may activate complement in vivo and impair host defenses by producing an opsonic defect for pneumococci. To examine this hypothesis, serum was incubated with suspensions of whole S. pneumoniae types 6, 7, 18, or 23 or with purified capsular polysaccharide from each of these types, and residual complement activity and opsonic capacity were measured. Hemolytic C 3--9 complement activity and opsonic capacity for 3H-thymidine labeled Salmonella typhimurium, a species effectively opsonized via the alternative pathway, were reduced in serum following incubation. Polysaccharide concentrations as low as 1 microgram/ml inhibited serum opsonic capacity for salmonella. Whole pneumococci and pneumococcal capsular polysaccharide also inhibited the opsonic activity of human C2 deficient serum for salmonella, further evidence for activation of complement via the alternative pathway. Pneumococcal capsular polysaccharide markedly inhibited the opsonic capacity of normal serum for the homologous pneumoccal type. Thus, amounts of pneumococcal capsular polysaccharide, similar to those found in the serum of patients with pneumococcal disease, bring about decomplementation of serum via activation of the alternative pathway and inhibit pneumococcal opsonization.     

C reactive protein concentrations during long distance running.

Long distance runners competing in events ranging from 15 to 88 km showed a distance related acute phase response as indicated by significantly raised serum C reactive protein concentrations. In trained athletes only a small rise in C reactive protein concentrations was seen after races of less than 21 km. After an 88 km ultramarathon concentrations comparable to those found in patients with small myocardial infarctions were detected. Indomethacin did not affect the increases in C reactive protein after the ultramarathon. This study has established serial C reactive protein concentrations for given race distances. These data may help in diagnosing myocardial infarction during long distance running. The acute phase response should be measured in untrained people running shorter distances to provide comparative data for the physically untrained population.     

Serum opsonic activity in rodent malaria: functional and immunochemical characteristics in vitro.

The functional and immunochemical characteristics of serum opsonic activity in rodent malaria were examined in the present study. Schizont- and late trophozoite-enriched populations of Plasmodium berghei-infected red blood cells (IRBC) were isolated on a Ficoll density-gradient and used in an in vitro phagocytosis system composed of serum and monolayer cultures of rat peritoneal macrophages. Hyperimmune serum augmented the phagocytosis of IRBC to a greater degree than did nonimmune serum. When either IRBC or macrophages were pre-incubated with serum, the phagocytosis-promoting factors acted on the IRBC rather than on the macrophages in a manner characteristic of serum opsonins. The opsonic activity was specific for IRBC since noninfected red blood cells were rarely phagocytized and were unable to absorb opsonic activity from serum. The opsonic activity of both hyperimmune and nonimmune sera was heat stable, and unaffected by agents known to inactivate or inhibit complement (cobra venom factor and ethylenediaminetetraacetic acid). Finally, the opsonic activity was identified in preparations of purified IgG isolated from both hyperimmune and nonimmune sera.     

THE ROLE OF REACTIVE OXYGEN SPECIES IN COPPER TOXICITY TO TWO FRESHWATER GREEN ALGAE1

The role of reactive oxygen species (ROS) in copper (Cu) toxicity to two freshwater green algal species, Pseudokirchneriella subcapitata (Korshikov) Hindák and Chlorella vulgaris Beij., was assessed to gain a better mechanistic understanding of this toxicity. Cu‐induced formation of ROS was investigated in the two algal species and linked to short‐term effects on photosynthetic activity and to long‐term effects on cell growth. A light‐ and time‐dependent increase in ROS concentrations was observed upon exposure to environmentally relevant Cu concentrations of 50 and 250 nM and was comparable in both algal species. However, effects of 250 nM Cu on photosynthesis were different, leading to a 12% reduction in photosynthetic activity in P. subcapitata, but not in C. vulgaris. These results indicate that differences in species‐specific sensitivities measured as photosynthetic activity were not caused by differences in the cellular ROS content of the algae, but probably by different species‐specific ROS defense systems. To investigate the role of ROS in Cu‐mediated inhibition of photosynthesis, the ROS scavenger N‐tert‐butyl‐α‐phenylnitrone (BPN) was used, resulting in a reduction of Cu‐induced ROS production up to control level and a complete restoration of photosynthetic activity of Cu‐exposed P. subcapitata. This finding implied that ROS play a primary role in Cu toxicity to algae. Furthermore, we observed a time‐dependent ROS release process across the plasma membrane. More than 90% of total ROS were determined to be extracellular in P. subcapitata, indicating an efficient method of cellular protection against oxidative stress.     

Variations in running activity and enzymatic adaptations in voluntary running rats

The running behavior and biochemical markers of oxidative and glycolytic activities associated with voluntary running activity were studied in male Sprague-Dawley rats after 6 wk of training in exercise wheel cages. Twenty-four-hour recordings of running activity were used to quantify the number of individual running bouts, their duration and running speed, and the distance run per day. We then established three categories of voluntary running activity based on the mean distance run per day during the last 3 wk of training: low-activity runners averaged 2-5 km/day, medium runners 6-9 km/day, and high runners greater than 11 km/day. Each group demonstrated an intermittent, nocturnal running pattern, at relatively high intensities, with a similar mean running speed for all groups (avg approximately 45 m/min). Differences in total distance run per day were the result of variations in both the number and duration of individual running bouts. Specifically, high runners (n = 7) had 206 +/- 30 individual running bouts per 24 h, each lasting 87 +/- 7 s; medium runners (n = 7) 221 +/- 22 running bouts, lasting 47 +/- 5 s; and low runners (n = 7) 113 +/- 7 bouts, each lasting 40 +/- 7 s. Voluntary running depressed the rate of body weight gain compared with sedentary control rats, despite an increased food and water intake for all runners. Furthermore, drinking activity was temporally associated with running periods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships of Age and Weekly Running Distance to BMI and Circumferences in 41, 582 Physically Active Women

Objective: To assess in women whether age‐related increases in adiposity are dependent on exercise, and, contrariwise, whether exercise‐related declines in adiposity are dependent on age. Research Methods and Procedures: Cross‐sectional analyses were conducted of 41, 582 female runners. Results: Age affected the relationships between vigorous exercise and adiposity. The decline in BMI per kilometer per week run was linear in 18 to 23 year olds and became increasingly non‐linear (convex) with age. Waist, hip, and chest circumferences declined significantly with running distance across all age groups, but the declines were significantly greater in older than younger women, particularly among shorter distance runners. The relationships between body circumferences and running distance became increasingly convex in older women. Conversely, vigorous exercise diminished the apparent increase in adiposity with age. The increase in average BMI with age was greatest in women who ran <8 km/wk, intermediate in women who ran 8 to 15 km/wk or 16 to 31 km/wk, and least in those who averaged over 32 km/wk. Before age 45, waist circumference rose for those who ran 0 to 7 km/wk, showed no significant relationship to age for those who ran 8 to 39 km/wk, and declined in those who ran 40 to 55 and 56 km/wk and more. Age related‐increases in hip and chest circumferences before 45 years old were significantly less in women who ran longer weekly distances. Discussion: These cross‐sectional associations are consistent with the hypothesis that exercise may mitigate age‐related increases in adiposity and that age affects exercise‐induced reductions in adiposity (although causality remains to be determined experimentally).     

Effect of opsonization on oxidative metabolism of plaice (Pleuronectes platessa L.) neutrophils

The effect of serum opsonization on Vibrio alginolyticus (heat-killed)-stimulated chemiluminescence (CL) by plaice kidney- and peritoneal exudate-derived neutrophils was investigated. Peritoneal neutrophils only recognized heat-labile and kidney neutrophils only heat-stable opsonic activity in normal serum. Specific antibody did not show opsonic activity nor any synergism with the normal serum opsonins for either neutrophil population. Evidence was found for the production, by plaice neutrophils, of H2O2, O2-, OH. and two or more, as yet unidentified, reactive oxygen species (ROS).     

The contribution of complement to opsonic activity in the uterine secretions of mares free of endometritis

The aim of the present study was to investigate if complement contributes to opsonic activity in the uterine secretions of mares with normal reproductive functions. Five mares with a mean age of 9 years were used in the study. The mares were considered to be free of endometritis based upon clinical history, palpation per rectum and ultrasonogaraphy of the genital tract, videoendoscopic inspection of the uterus, electronmicroscopy of endometrial biopsies, and bacteriological and cytological examination of swabs from the endometrium. The hormonal status of the mares was also determined. Uterine secretions were collected by a tampon inserted into the uterus during estrus. Secretion and serum samples were divided in half, one half of which was subjected to heat treatment (56 degrees C in 30 minutes) in order to inactivate the complement. Opsonic activity in the samples was then determined in a chemiluminescence assay using zymosan as the target particle. Opsonic activity was expressed as peak chemiluminescence and the time to peak chemiluminescence. Heat treatment of the secretions reduced peak luminescence significantly (P<0.01), and it prolonged the time to peak luminescence significantly (P<0.05). These data show that complement contributes a considerably to opsonic activity in uterine secretions prior to an inflammatory response in the equine uterus.     

The method for estimation of opsonic activity in sera of infants

Three methods for the estimation of opsonic activity in the sera of newborn children were tested. Two of them, based on the phagocytosis of opsonised bacteria labelled with radioactive phosphorus³²P as measured byin vivo blood clearance or uptake of bacteria in perfused isolated liver, were found to be unsuitable for long term dynamic study mainly because they do not permit the testing of series of samples. The third method (using isolated phagocytic cellsin vitro) permits the differentiation of the opsonic effect of complement and antibody and, furthermore, the firmness of the bond between microbes and phagocytes (which reflects the degree of opsonization) can be established. It was found that a 2-mercaptoethanol-resistant antibody, probably of the IgG type, was responsible for the opsonic activity of children's sera toEscherichia coli 083. Homologous antibody (toEscherichia coli 083) could be differentiated from beterologous antibody (toEscherichia coli 086) using the opsonic test only at low dilutions of sera. The combination of newborn piglet complement and antibody of children's sera yielded higher values of opsonic activity than either component separately.     

Calcium as a possible modulator of Kupffer cell phagocytic function by regulating liver-specific opsonic activity

Recently we described some properties of organ-specific serum opsonins which differentiate between liver- and spleen-specific opsonic activities, and reported that, on dialysis of serum, its liver opsonic activity is enhanced by 2- to 3-fold, whereas spleen-specific activity is reduced by 20-30% of that of control serum (Moghimi, S.M. and Patel, H.M. (1989) Biochim. Biophys. Acta 984, 379-383). This observation suggests that serum contains dialysable factors which regulate liver- as well as spleen-specific opsonic activities. Our results from EGTA-treated serum suggest that dialysable factor(s) could be divalent cations such as Ca2+, Mn2+, Mg2+ or Co2+, and among them, calcium may be the key regulatory factor for liver-specific opsonic activity. The regulatory mechanism of spleen-specific opsonic activity seems to be complex, since addition of dialysate or calcium or magnesium to the dialysed serum does not restore its activity; probably the removal of divalent cations has induced an irreversible conformational change in spleen-specific opsonin. In conclusion, we propose that the blood calcium concentration may play an important role in modulating hepatic phagocytic function by modifying liver-specific opsonic activity in serum. An increase in the physiological concentration of calcium will suppress and a decrease will enhance this opsonic activity.     

Exploitation of the complement system by Leishmania promastigotes

Complement has long been acknowledged as an important component of host defense to extracellular pathogens. David Mosser and Andrew Brittingham here describe how Leishmania spp, intracellular pathogens of mononuclear phagocytes, exploit the opsonic and chemotactic effects of complement to initiate infection in the mammalian host.     

Role of complement component C1q in the IgG-independent opsonophagocytosis of group B streptococcus.

We investigated the role of complement component C1q in the IgG-independent opsonophagocytosis of type III group B Streptococcus (GBS) by peripheral blood leukocytes. We report that C1q binds to type III GBS both in normal human serum deficient in IgG specific for type III capsular polysaccharide and in a low-ionic strength buffer. The dissociation constant Kd ranged from 2.0 to 5.5 nM, and the number of binding sites Bmax ranged from 630 to 1360 molecules of C1q per bacterium (CFU). An acapsular mutant strain of GBS bound C1q even better than the wild type, indicating that the polysaccharide capsule is not the receptor for C1q. In serum, binding of C1q to GBS was associated with activation of the classical complement pathway. However, normal human serum retained significant opsonic activity after complete depletion of C1q, suggesting that the serum contains a molecule that is able to replace C1q in opsonization and/or complement activation. Mannan-binding lectin, known to share some functions with C1q, appeared not to be involved, since its depletion from serum had little effect on opsonic activity. Excess soluble C1q or its collagen-like fragment inhibited phagocytosis mediated by normal human serum, suggesting that C1q may compete with other opsonins for binding to receptor(s) on phagocytes. We conclude that, although C1q binds directly to GBS, C1q binding is neither necessary nor sufficient for IgG-independent opsonophagocytosis. The results raise the possibility that additional unknown serum factor(s) may contribute to opsonization of GBS directly or via a novel mechanism of complement activation.